The goal of this proposal is to investigate the role of protein motions in the catalytic function of the MAP kinase, ERK2, and to define how these dynamics can be used to evaluate inhibitors towards this important therapeutic target. ERK1/2 protein kinases are essential for a wide range of fundamental processes, including cell cycle progression and proliferation, cell movement and adhesion, and cell survival. Because they underlie many human diseases, they are crucial targets for therapeutics. However, clinically effective inhibitors of these enzymes have not been found yet. Our study uses NMR relaxation dispersion measurements to demonstrate that phosphorylation and activation of ERK2 induces dramatic changes in the dynamics of the enzyme, leading to global conformational exchange. From this we have developed a new hypothesis that the activation of ERK2 involves release of a thermodynamic barrier to dynamics, thus enabling conformational exchange and motions that accompany the formation of enzyme intermediates during progression through the catalytic cycle. We propose that exploiting these features of protein dynamics will improve inhibitor design for this key enzyme. The objective of the proposal is to investigate the hypothesis that protein dynamics plays an important role in the activation mechanism and catalytic cycle of ERK2, and determine how this mechanism impacts the behavior of inhibitors towards this important therapeutic target. The Specific Aims will address three fundamental goals: Aim 1. Determine the energy profile for the kinase reaction cycle in ERK2. This aim will identify which steps in the kinase reaction are controlled by protein motions, by combining NMR CPMG and 2D-TROSY experiments with measurements of individual rate constants for each step in the catalytic cycle. Aim 2. Determine the role of conformational exchange on the properties of ERK inhibitors. This aim will compare small molecule inhibitors and examine structure-activity relationships to test the hypothesis that ERK2 dynamics enables conformational selection which confers inhibitors with favorable kinetic properties. Aim 3. Develop a structural model for the L and R conformers. This aim will determine structures of ligand-bound complexes of 2P-ERK2 by X-ray crystallography that we propose represent the R conformer, and examine structural elements that form the high thermodynamic barrier to LR conversion in 0P-ERK2. Our interdisciplinary approach integrates technologies of NMR relaxation, X-ray crystallography, pre-steady state and single turnover enzyme kinetics, and inhibitor structure-activity relationships. The outcomes of this proposal will establish ERK2 as a new experimental model to investigate the role of protein dynamics in kinase activation and catalysis, and determine the importance of this mechanism for inhibitor design.